Synthesis and Fluorescent Properties of Alkynyl- and Alkenyl-Fused Benzotriazole-Derived α-Amino Acids

Fluorescent unnatural α-amino acids are widely used as probes in chemical biology and medicinal chemistry. While a variety of structural classes have been developed, there is still a requirement for new environmentally sensitive analogues that can closely mimic proteinogenic α-amino acids. Here, we report the synthesis and fluorescent properties of highly conjugated, benzotriazole-derived α-amino acids designed to mimic l-tryptophan. Alkynyl-substituted analogues were prepared using three key steps, nucleophilic aromatic substitution with a 3-aminoalanine derivative, benzotriazole formation via a one-pot diazotization and cyclization process, and a Sonogashira cross-coupling reaction. E-Alkenyl-substituted benzotriazoles were accessed by stereoselective partial hydrogenation of the alkynes using zinc iodide and palladium catalysis. The alkynyl analogues were found to possess higher quantum yields and stronger brightness and, a solvatochromic study with the most fluorogenic α-amino acids demonstrated sensitivity to polarity.


■ INTRODUCTION
Fluorescence spectroscopy has become a powerful technique for the investigation of biological structure and function, and for visualizing cellular processes at the molecular level. 1 In combination with the advances in fluorescent-based technology, libraries of small-molecule probes containing chromophores that are tuned for specific applications have been reported. 2 As peptides and proteins are important for a wide range of biological processes, there has been significant interest in the discovery of fluorescent, unnatural α-amino acids that can be specifically incorporated into a protein, while retaining the original structure and function. 3 A major strategy in the development of novel, fluorescent α-amino acids has been the structural modification of L-tryptophan (1), the most fluorescent proteinogenic α-amino acid ( Figure 1). To improve the intrinsic optical properties of the indole side chain and to prevent spectroscopic overlap with L-tryptophan residues already present in a protein, studies have focused on the preparation of analogues with extended conjugation. 4 For example, cyanotryptophans have proven to be excellent structural analogues of L-tryptophan, with significantly improved optical properties. 5 L-6-Cyanotryptophan has been used as a fluorescent probe for studying protein conforma-tional changes, 6 while the blue fluorescent amino acid L-4cyanotryptophan (2) has been used to assess peptide− membrane interactions. 7 Tryptophan compounds with extended conjugation at the C-2 position of the indole have also been prepared via coupling reactions with triazoles or arenes. 8 This general approach has been used by Vendrell and coworkers, who have developed a series of L-tryptophan analogues substituted at the C-2 position with BODIPY chromophores. 9,10 This includes L-tryptophan-BODIPY conjugate 3 that was incorporated into a cyclic peptide and used for the visualization of fungal infections in human tissue. 9 We have previously reported the synthesis and fluorescent properties of various classes of α-amino acids, 11 including the preparation of benzotriazole-derived α-amino acids, as potential structural mimics of L-tryptophan. 12 In a previous study, a Suzuki−Miyaura cross-coupling reaction was used to extend the conjugation of the benzotriazole side chain allowing access to 5-aryl analogues such as compound 4 ( Figure 1). Incorporation of various electron-rich arenes generated fluorescent amino acids with MegaStokes shifts. However, the main absorption band of these compounds was similar to that of proteinogenic α-amino acids, such as L-tyrosine and Ltryptophan, and thus, prohibited the use of these as fluorescent probes in proteins containing these residues. To overcome this limitation, we were interested in the development of new analogues with extended conjugation that would exhibit absorption at longer wavelengths. Here, we report the design and synthesis of a new class of fluorescent α-amino acid that incorporates an alkynyl or alkenyl spacer unit between the benzotriazole and aryl groups. We also describe the fluorescent properties of these compounds and demonstrate that as well as possessing absorption at a longer wavelength, key analogues from the alkynyl series are brighter and highly sensitive to environment polarity.

■ RESULTS AND DISCUSSION
As shown in Scheme 1, our proposed approach to α-amino acids bearing alkynyl-and alkenyl-substituted benzotriazole side chains involved the synthesis of 5-iodobenzotriazole 8 as a key intermediate. The planned three-step preparation of 8 involved a nucleophilic aromatic substitution reaction of 2fluoro-5-iodonitrobenzene (6) with 3-aminoalanine derivative 5. Following reduction of the nitro group, a one-pot diazotization and cyclization reaction under mild conditions would yield 5-iodobenzotriazole 8. Sonogashira coupling of 8 with a range of aryl-substituted alkynes would result in the preparation of the alkynyl targets. Stereo-and chemoselective reduction of the alkynyl compounds would then allow the rapid synthesis of the second set of targets, the styryl analogues.
Initially, gram quantities of N-Boc-L-3-aminoalanine αmethyl ester 5 were prepared from commercially available N-Boc-L-asparagine 9 as previously described by Piantanida and co-workers (Scheme 2). 13 The four-step route that involved a Hofmann rearrangement and protecting group manipulation gave 5 in 69% overall yield. Nucleophilic aromatic substitution of 3-aminoalanine derivative 5 with 2-fluoro-5-iodonitrobenzene (6) using triethylamine gave adduct 10 in 78% yield. Chemoselective nitro group reduction of 10 was then performed using zinc and acetic acid. Reduction under mild acidic conditions and with a short reaction time of 0.75 h allowed full conversion, while maintaining both the C−I bond and Boc-group protection of the amine. This gave aniline 7 in 91% yield. The one-pot activation of the amine as the diazo intermediate and in situ cyclization to benzotriazole 8 was performed as previously described by us. 12,14 Formation of the diazo intermediate was achieved under mild conditions, using a polymer-supported nitrite reagent and p-tosic acid. 15,16 The one-pot reaction was complete after 1 h and gave benzotriazole 8 in 73% yield. Again, the integrity of the Boc-protecting group was not affected by the acidic conditions. The synthesis of the α-amino acids with extended alkynyl-fused benzotriazole chromophores was then achieved using a Sonogashira reaction of iodobenzotriazole 8 with various electron-rich and electrondeficient aryl-substituted acetylenes. Under standard conditions, this gave the coupled products 11a−f in 70−95% yields. 17,18 Crucial to the success of the coupling reactions was the requirement of an initiation phase at high temperature (100°C for 0.1 h), before conducting the remainder of the reaction at room temperature. Initial heating allows efficient formation of the active palladium(0) species from the precatalyst, while returning to room temperature for the coupling step prevents extensive Glaser−Hay coupling of the terminal alkyne reagent. 19 Deprotection to the parent α-amino acids was then performed using a two-step approach. Ester hydrolysis with cesium carbonate was followed by the removal of the Boc-group under acidic conditions. Despite the presence of electron-rich alkynes, acidic deprotection proceeded cleanly, without any significant byproducts. Purification by recrystallization gave the amino acid hydrochloride salts 12a−f in good overall yields.
To access the corresponding alkenyl analogues, various approaches were attempted. These included the Heck crosscoupling reaction of styrene with iodobenzotriazole 8 and although successful, competing protodepalladation during the reaction resulted in a low yield of the desired product (32%). A second attempt involved the Suzuki−Miyaura cross-coupling of 2-arylvinylboronic acids with a bromo-analogue of 8. However, this again gave the alkenyl-coupled benzotriazole in low yield (25%). For these reasons, the reduction of the alkynyl-fused benzotriazoles 11 was considered as a possible approach to the corresponding alkenyl analogues. From the range of literature methods available, a chemo-and stereoselective hydrogenation procedure at atmospheric pressure, reported by Jackowski and co-workers, was considered. 20 This reaction involves the combination of a palladium catalyst and zinc(II) iodide, which promotes syn-hydrogenation, followed by Z-to E-isomerization. Attempted reduction of alkyne 11b using the standard conditions (5 mol % of Pd cat. and 25°C) required a reaction time of 112 h and gave a 2:1 mixture of Eand Z-alkenes isomers (Table S1). Optimization studies showed that by increasing both the amount of palladium catalyst (20 mol %) and reaction temperature (40°C), the reaction time for reduction of 11b could be reduced to 20 h, to give solely E-isomer 13b in 64% yield (Scheme 3). Having The Journal of Organic Chemistry pubs.acs.org/joc Article prepared an electron-rich analogue, an electron-neutral analogue 11a, and an electron-deficient analogue 11e were also subjected to the optimized reduction conditions. In both cases, the reaction generated only the E-isomer (13a and 13c) in similar yields. Ester hydrolysis with cesium carbonate and mild acid removal of the Boc-group gave the deprotected amino acids in high overall yields. Following the synthesis of the alkynyl-and alkenyl-fused benzotriazole-derived α-amino acids, the optical properties were measured for each compound and compared with 5-aryl analogue 4 (Figures 2 and 3, and Table 1). 12,21 The ultraviolet/visible (UV/visible) absorption and photoluminescence spectra of the α-amino acids were recorded in methanol at a concentration of 5 μM. As proposed, the extended chromophores all displayed red-shifted absorption. Direct comparison of the p-methoxy analogues shows absorption bands at 321 nm for alkyne 12b and 320 nm for alkene 14b vs 256 nm for 5-aryl analogue 4. Strong fluorescence with emission maxima in the visible region was observed for benzotriazoles containing electron-rich aryl side chains. 22 In addition, the alkynyl series of compounds displayed the most favorable properties. For example, p-methoxyphenyl 12b possessed a quantum yield of 34% and was significantly brighter than both structural analogues 4 and 14b. These results suggest that the aryl-substituted alkynyl-benzotriazoles can readily adopt a flat conformation which allows effective conjugation across the chromophore, compared to the biaryl   (4) and alkenyl (14b) systems that can relax via nonradiative decay pathways due to extra rotational and vibrational modes. 23 In addition to the strong fluorescence of electronrich analogues, naphthyl-substituted alkyne 12d possessed the highest quantum yield (42%) and good brightness. Overall, the strategy of inserting alkynyl and alkenyl spacer units to extend the chromophores has yielded compounds with improved photophysical properties, particularly absorption maxima that no longer overlap with proteinogenic amino acids. It should be noted that while this has resulted in compounds with smaller Stokes shifts than the original series, these are still significantly large (e.g., 7400 cm −1 for 12b and 8677 cm −1 for 14b).
As the p-methoxyphenyl-and naphthyl-substituted alkynylfused benzotriazoles 12b and 12d were found to be the brightest α-amino acids, the properties of these compounds were further explored via a solvatochromic study. 24 In contrast to the absorption bands of both compounds, which were found to be independent of solvent polarity (see the Supporting Information), the emission maxima displayed significant bathochromic shift with increasing polarity (Figure 4). For example, benzotriazole 12b displayed an emission maximum at 367 nm in ethyl acetate compared to 460 nm in water. Similarly, benzotriazole 12d showed a range from 348 to 426 nm. The solvatochromism exhibited by 12b and 12d suggests that the excited state has internal charge transfer character, which is stabilized in more polar solvents. The larger bathochromic shift of 12b implies a stronger dipole across the chromophore, which would be expected for a more electron-rich substituent. The solvatochromism for 12b and 12d was further evidenced from Lippert−Mataga plots, in which graphs of Stokes shifts versus solvent orientation polarizability showed a linear correlation (see the SI). 25 The linearity of these plots confirms the general effect of solvent in the shift of emission bands. Both the bathochromic shift of emission in aqueous solvents and the environment sensitivity of these amino acids suggests potential as probes for chemical biology applications.

■ CONCLUSIONS
In summary, a series of alkyne-fused benzotriazole-derived αamino acids have been prepared using nucleophilic aromatic substitution with a 3-aminoalanine derivative, a one-pot diazotization, and cyclization to access the benzotriazole unit and a Sonogashira cross-coupling reaction for the introduction of the unsaturated side chain as the key steps. Access to the corresponding E-alkenes was achieved by a chemo-and stereoselective, palladium-catalyzed hydrogenation reaction. Investigation of the photophysical properties of both classes of α-amino acids revealed that extended conjugation resulted in compounds with red-shifted absorption bands. This means these compounds can be excited in the presence of fluorescent proteinogenic α-amino acids. The majority of compounds demonstrated strong fluorescent properties and large Stokes shifts, with the alkyne series possessing the highest quantum yields and brightest chromophores. A solvatochromic study with the brightest α-amino acids showed significant environment sensitivity to solvent polarity. Work is currently underway to investigate the use of compounds 12b and 12d as probes in chemical biology applications.

■ EXPERIMENTAL SECTION
The synthesis of compound 5 has been previously described in the literature. 13 All reagents and starting materials were obtained from commercial sources and used as received. Reactions were performed open to air unless otherwise mentioned. All reactions performed at elevated temperatures were heated using an oil bath. Brine refers to a saturated aqueous solution of sodium chloride. Flash column chromatography was performed using silica gel 60 (40−63 μm). Aluminum-backed plates precoated with silica gel 60F 254 were used for thin-layer chromatography and were visualized with a UV lamp or by staining with potassium permanganate, vanillin, or ninhydrin. 1 H NMR spectra were recorded on an NMR spectrometer at either 400 or 500 MHz and data are reported as follows: chemical shift in ppm relative to the solvent as internal standard (CHCl 3 , δ 7.26 ppm; CH 3 OH, δ 3.31 ppm; DMSO, δ 2.50), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or overlap of nonequivalent resonances, integration). The abbreviations br s and br d refer to broad singlet and broad doublet, respectively. 13 C NMR spectra were recorded on an NMR spectrometer at either 101 or 126 MHz and data are reported as follows: chemical shift in ppm relative to tetramethylsilane or the solvent as internal standard (CDCl 3 , δ 77.2 ppm; CD 3 OD, δ 49.0 ppm; DMSO-d 6 , δ 39.5), multiplicity with Table 1. Photophysical Data of Benzotriazole-Derived α-Amino Acids Spectra were recorded at 5 μM in methanol. b Quantum yields (Φ F ) were determined in methanol using anthracene and L-tryptophan as standards. Emission spectra of 12d in various solvents. All spectra were recorded using a concentration of 5 μM.
respect to hydrogen (deduced from DEPT experiments, C, CH, CH 2 or CH 3 ). Infrared spectra were recorded on a Fourier transform infrared (FTIR) spectrometer; wavenumbers are indicated in cm −1 . Mass spectra were recorded using electrospray techniques. Highresolution mass spectra (HRMS) were recorded using quadrupole time-of-flight (Q-TOF) mass spectrometers. Melting points are uncorrected. Optical rotations were determined as solutions irradiating with the sodium D line (λ = 589 nm) using a polarimeter.

* sı Supporting Information
The